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Dermaseptin B1 (DrsB1), an antimicrobial cationic 31 amino acid peptide, is produced by Phyllomedusa bicolor. In an attempt to enhance the antimicrobial efficacy of DrsB1, the DrsB1 encoding 93 bp sequence was either fused to the N or C terminus of sequence encoding chitin‐binding domain (CBD) of Avr4 gene from Cladosporium fulvum. Tobacco leaf disk explants were inoculated with Agrobacterium rhizogenes harboring pGSA/CBD‐DrsB1 and pGSA/DrsB1‐CBD expression vectors to produce hairy roots (HRs). Polymerase chain reaction (PCR) was employed to screen putative transgenic tobacco lines. Semi‐quantitative RT‐PCR and western blotting analysis indicated that the expression of recombinant genes were significantly higher, and recombinant proteins were produced in transgenic HRs. The recombinant proteins were extracted from the tobacco HRs and used against Pectobacterium carotovorum, Agrobacterium tumefaciens, Ralstonia solanacearum, and Xanthomonas campestris pathogenic bacteria and Alternaria alternata and Pythium sp. fungi. Two recombinant proteins had a statistically significant (p < 0.01) inhibitory effect on the growth and development of plant pathogens. The CBD‐DrsB1 recombinant protein demonstrated a higher antibacterial effect, whereas the DrsB1‐CBD recombinant protein demonstrated greater antifungal activity. Scanning electron microscopy images revealed that the structure of the fungal mycelia appeared segmented, adhered to each other, and crushed following the antimicrobial activity of the recombinant proteins. Due to the high antimicrobial activity of the recombinant proteins against plant pathogens, this strategy can be used to generate stable transgenic crop plants resistant to devastating plant pathogens.

Antimicrobial peptides have been long known to confer resistance to plant pathogens. In this study, new recombinant peptides constructed from a dermaseptin B1 (DrsB1) peptide fused to a chitin-binding domain (CBD) from Avr4 protein, were used for Agrobacterium tumefaciens-mediated transformation of tobacco plants. Polymerase chain reaction (PCR), semi-quantitative RT-PCR, and western blotting analysis demonstrated the incorporation and expression of transgenes in tobacco genome and transgenic plants, respectively. In vitro experiments with recombinant peptides extracted from transgenic plants demonstrated a significant (P<0.01) inhibitory effect on the growth and development of plant pathogens. The DrsB1-CBD recombinant peptide had the highest antifungal activity against fungal pathogens. The expression of the recombinant peptides greatly protected transgenic plants from Alternaria alternata, Alternaria solani, Fusarium oxysporum, and Fusarium solani fungi, in comparison to Pythium sp. and Pythium aphanidermatum. Expression of new recombinant peptides resulted in a delay in the colonization of fungi and appearance of fungal disease symptoms from 6 days to more than 7 weeks. Scanning electron microscopy images revealed that the structure of the fungal mycelia appeared segmented, cling together, and crushed following the antimicrobial activity of the recombinant peptides. Greenhouse bioassay analysis showed that transgenic plants were more resistant to Fusarium and Pythium infections as compared with the control plants. Due to the high antimicrobial activity of the recombinant peptides against plant pathogens and novelty of recombinant peptides, this report shows the feasibility of this approach to generate disease resistance transgenic plants.

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